The BCHE and ACHE genes encoding the acetylcholine hydrolyzing enzymes butyrylcholinesterase (BuChE, EC 3.1.1.8) and actylcholinesterase (AChE, EC 3.1.1.7) are expressed in various developing cell types, including embryonic Layer, P. G. and Sporns, O., Proc. Natl. Acad. Sci. USA 84:284-288 (1987)!, hematopoietic Burstein, S. A., et al., J. Cell Physiol. 122:159-165 (1985)! and germ cells Johnson, C. D., et al., Neuron 1:165-173 (1988); Malinger, G., et al., Mol. Neurosci. 1:77-84 (1989)!.
Both AChE and BuChE include the peptide motif S/T-P-X-Z, which makes-them potential substrates for phosphorylation by cdc2 kinases, the general controllers of the cell cycle Lapidot-Lifson, Y., et al., Proc. Natl. Acad. Sci., USA 89: 579-583 (1992)!. Most other substrates of cdc2 kinases perform biological functions necessary for cell cycle-related processes Moreno, S. and Nurse, P., Cell 61:549-551 (1990)!. Thus, interference with either CHE or cdc2 transcription processes may be expected to divert and/or arrest cell division, and controlling these processes can be useful for several, medically important, procedures.
Biochemical and histochemical analyses indicate that both AChE and BuChE are expressed, in high levels, in various fetal tissues of multiple eukaryotic species Rakonczay, Z., et al., Subcellular Biochemistry 12:335-378, Harris, J. R., Ed., Plenum Press, N.Y. (1988)!, where cholinesterases (ChEs) are coordinately regulated with respect to cell proliferation and differentiation Layer, P. G., et al., Neurochem. 49:175-182 (1987)!. The specific role to be attributed to ChEs in embryonic development may hence be related with cell division, so that their biological function(s) in these tissues are tentatively implicated in the control of organogenesis.
In addition to its presence in the membranes of mature erythrocytes, AChE is also intensively produced in developing blood cells in vivo Paulus, J. P., et al., Blood 58:1100-1106 (1981)! and in vitro Burstein, S. A., et al., J. Cell Physiol. 103:201-208 (1980)! and its activity serves as an acceptable marker for developing mouse megakaryocytes Burs- tein (1985) ibid.!. Furthermore, administration of acetyl- choline analogues as well as cholinesterase inhibitors has been shown to induce megakaryocytopoiesis and increased platelet counts in the mouse Burstein, S. A., et al., Clin. Haematol. 12:3-27 (1983)!, implicating this enzyme in the commitment and development of these haematopoietic cells.
The DNAs coding for human BuChE and AChE have been cloned Prody, C., et al., Proc. Natl. Acad. Sci., USA 86:3555-3559 (1987); Soreq et al., Proc. Natl. Acad. Sci., USA 87:9688-9692 (1990)! and the human CHEl locus has been mapped Gnatt, A., et al., Cancer Res. 50:1983-1987 (1990)! to the 3q26-ter chromosomal domain that is subject to aberrations in leukemias accompanied by abnormal megakaryocytopoiesis and platelet counts Pintado, T., et al., Cancer 55:535-541 (1985)!. Co-amplification of the ACHE and BCHE genes was subsequently observed in leukemias and platelet disorders Lapidot-Lifson, Y., et al., Proc. Natl. Acad. Sci., USA 4715-4717 (1989); Zakut, H., et al., Mutation Research, in press (1992)!. The hemopoietic system thus appears to be subject to developmental control as affected by the expression of the ChEs.
Inhibition of the expression of developmentally important genes should, in principle, divert developmental processes to directions which are not dependent on the expression of these genes. One useful approach to affect such developmental processes is based on "antisense" technology. The use of oligonucleotides to intervene in the genetic processes of the cell bears an important therapeutic potential. Thus, "informational drugs" with possible clinical applications are being developed which arrest the expression of cloned genes. The hematopoietic system may be the first logical target for novel therapy protocols based on the recent achievement in genetic engineering, since it includes proliferating stem cells and because of its extreme sensitivity to external stimuli Wilson, J. D., et al., Harrison's Principles of Internal Medicine, 12th Ed., McGraw-Hill, Inc., New York, Chapters 268-269; 285-288 (1991)!. Stem cells may be defined as cells which can replicate repeatedly and differentiate into various kinds of committed cells. Commitment will gradually limit the differentiation choices for cells in which it occurs, until precursor cells are formed with only one choice (i.e. erythrocytes, megakaryocytes or macrophages). The first stem cells can thus be defined as totipotent, i.e. they may make all of the choices in the blood and immune system. The orientation of such cells into desired direction should be most useful in overcoming undesired changes, such as depletion or excess of specific subpopulations of hemopoietic cells. Less, although also useful is the redirection of the more limited pluripotent stem cells. Stem cells account for .ltoreq.0.1% of cells in bone marrow. They can be detected either directly, by immunocytochemical methods, or retroactively, by cell culture growth and subsequent evaluation of formed colonies. For therapeutic purposes, it would be desirable to control stem cells differentiation and cause totipotent and pluripotent stem cells to replicate.
Production rate of bone marrow cells in healthy individuals may reach 10.sup.10 platelets and differentiated blood cells per hour. Life span of these cells varies from years for some lymphocytes, 120 days for erythrocytes to 10 days for platelets and 10 hours for neutrophils. Changes in the subpopulations of hemopoietic stem cells may be found in patients suffering malignant myeloproliferative diseases, such as various leukemias etc., in blood cells proliferative diseases such as polycythemia vera etc., and in autoimmune diseases like lupus erythomatosus etc., in which the blood production system is defective. Defective hemopoiesis is further observed in patients undergoing various commonly used therapeutical treatments like chemotherapy and irradiation which impair the blood production system. Thus all cancer patients, individuals following tissue transplantation and others who have suffered poisoning by chemicals and/or different drugs, display abnormal hemopoiesis with its subsequent consequences. The therapeutical value of the ability to modulate hemopoietic cell division in patients suffering any of the above pathological conditions is self-evident. Furthermore, modulation of hemopoietic cell division, and especially increasing the number of hemopoietic stem cells, may be of particular advantage for the clinical procedure of bone marrow transplantation. Techniques are already available for freezing bone marrow cells and for their subsequent transplantation in patients suffering any of the above pathological conditions. However, the only transplanted bone marrow cells which can survive in the recipient and proliferate to improve his/hers condition are stem cells. The above procedure may be autotransplantation, using the recipient's own bone marrow, or allotransplantation, using bone marrow from a compatible donor.
As mentioned above, cholinergic signals are implicated in the commitment and development of haematopoietic cells. Recently, preliminary evaluation of antisense oligonucleotides incorporation in vivo was performed which revealed the short term therapeutic applicability of this approach Cohen, et al., Antisense Res. & Dev. 2:191 (1991)!. Antisense oligonucleotides of 15-20 bases are usually long enough to ascertain that they will only have one complementary sequence in the mammalian genome. In addition, they hybridize well with their target mRNA Cohen et al., ibid.!. Modification of the phosphodiester backbone renders these oligonucleotides resistant to degradation by nucleases Spitzer, F. and Eckstein, F., Nuc. Ac. Res. 16: 11691-11704 (1988)!. Both methylphosphonate and phosphorothioate groups were used for this purpose Baker, C., et al., Nuc. Ac. Res. 18:3537 (1990)!. Being nonionic, the methyl-phosphonate analogs were predicted to exhibit increased cellular uptake Blake, et al., Biochem. 24:6139 (1985)!. However, antisense methylphosphonate oligomers were shown to be incapable of inhibiting N-ras expression in vitro Tidd, et al., Anti-Cancer Drug Design 3:117 (1988)! whereas the in vitro translation of several oncogene mRNAs was successfully blocked by phosphodiester and/or phosphorothioate antisense oligonucleotides c-myc: McManaway et al., Lancet 335:808 (1990), Watson et al., Cancer Res. 51:3996 (1991); bcl-2: Reed et al., Cancer Res. 50:6565 (1990); myb: Calabrett et al., Proc. Natl. Acad. Sci. USA 88:2351 (1991); bcr-ab: Szczylik et al., Science 253:562 (1991)!. Both sense and nonsense oligonucleotides served as controls in these studies and were shown to be non-effective, while antisense oligonucleotides selectively inhibited their target gene expression and phosphorothioate oligonucleotides were more potent because of their greater stability Woolf, T. M., et al., Nuc. Ac. Res. 18:1763 (1990)3.
Antisense oligonucleotides are able to interfere specifically with synthesis of the target protein of interest Moffat, Science 253:510 (1991)!. This may occur by inhibition of polysome formation and/or functioning, according to the position of the antisense oligonucleotide within the target mRNA. Thus, the frequent choice of the sequence surrounding the translation initiation codon as target for antisense oligonucleotide inhibition aims to prevent the formation of the initiation complex. Indeed, antisense RNAs occur naturally as regulators of translation Eguchi et al., Ann. Rev. Biochem. 60:631 (1991)!. Other mechanisms of antisense oligonucleotide inhibition involve activation of ribonuclease H, which subsequently performs digestion of the antisense oligonucleotide-mRNA hybrids Chiang, M. Y., et al., J. Biol. Chem. 266:18162 (1991)!, or interference with splicing through antisense oligonucleotides targeted to mRNA splice sites Kole et al., Adv. Drug Deliv. Rev. 6:271 (1991)!.
In addition to their mRNA targets, antisense oligonucleotides are also complementary to the genomic sequences expressing these mRNAs. When injected into cultured cells, they accumulate within nuclei Leonetti, J. P., et al., Proc. Natl. Acad. Sci. USA 88:2702 (1991)!, suggesting that they may also function by interfering with transcription through formation of a third DNA strand, associated by Hoogsteen base pairing with the major groove of the B-form DNA duplex Moffat, Science 252:1374 (1991)!. In vitro transcriptional arrest of c-myc expression was shown to operate by this mechanism in a cell-free system cooney et al., Science 241: 456 (1988)!. Recent polyamide nucleic acid oligomers (with polyamide backbone replacing the deoxyribose phosphate backbone of DNA) were shown to cause displacement of their complementary strands from double stranded DNA Nielsen et al., Science 254:1497 (1991)!. These newly designed drugs will selectively interrupt gene function without affecting the transcript products. In contrast, ribozyme sequences were shown to specifically interact with the mRNA transcripts. These are ribonucleic acid sequences, including RNase active sites flanked by antisense oligonucleotides Haseloff and Gerlach, Nature 3:585 (1988)!. When targetted to the human immunodeficiency virus (HIV) they destroy HIV mRNA effectively Sarver et al., Science 247:1222 (1990)!. However, oligoribonucleotides are more difficult to synthesize than oligodeoxynucleotides, particularly in chemically modified forms resistant to RNase attacks Pieken et al., Science 253:314 (1991)!.
Phosphorothioate antisense oligonucleotides do not show significant toxicity and exhibit sufficient pharmacodynamic half-lives in animals Agrawal, s., et al., Proc. Natl. Acad. Sci. USA 88:7595 (1991)!. Antisense induced loss-of-function phenotypes related with cellular development were shown for the glial fibrillary acidic protein (GFAP), implicated in astrocyte growth within astrocyte-neuron cocultures Winstein et al., J. Cell Biol., 112:1205 (1991)!, for the myelin-associated glycoprotein in Schwann cells, responsible for formation of the compact myelin sheath formation surrounding these cell Owens and Bunge, Neuron 7:56 (1991)!, for the microtubule-associated tau proteins implicated with the polarity of hippocampal neurons and their axon formation Caceres and Kosik, Nature 343:461 (1990)!, for the .beta..sub.1 -integrin, important for neuronal migration along radial glial cells, and for the establishment of tectal plate formation in chick Galileo et al., J. Cel. Biol. 112:1285 (1991)! and for the N-myc protein, responsible for the maintenance of cellular heterogeneity in neuroectodermal cultures (ephithelial vs. neuroblastic cells, which differ in their colony forming abilities, tumorigenicity and adherence) Rosolen et al., Cancer Res. 50:6316 (1990); Whitesell et al., Mol. Cell. Biol 11:1360 (1991)!. Antisense oligonucleotide inhibition of basic fibroblast growth factor (bFgF), having mitogenic and angiogenic properties, suppressed 80% of growth in glioma cells Morrison, J. Biol. Chem. 266:728 (1991)! in a saturable and specific manner. The antisense oligonucleotides were targetted against the initiation and splice sites in bFgFmRNA, they reduced activity of the resulting protein and sense oligomers remained inactive. In soft-agar cultures, antisense oligonucleotides reduced the size of glial colonies and induced appearance of larger cells within them R. Morrison, Neuroscience Facts 3: 3 (1992): bFGF expression in human glioma cells)!.
Being hydrophobic, antisense oligonucleotides interact well with phospholipid membranes Akhtar, S., et al., Nuc. Res. 19:5551-5559 (1991)!. Following their interaction with the cellular plasma membrane, they are actively transported into living cells (Loke, S. L., et al., Proc. Natl. Acad. Sci. USA 86:3474 (1989)!, in a saturable mechanism predicted to involve specific receptors Yakubov, L. A., et al., Proc. Natl. Acad. Sci. USA 86:6454 (1989)!.
Antisense inhibition of key molecules involved in signal transduction processes may De expected to interfere also with secondary mechanisms depending on the targetted key molecule. Thus, cholinergic signaling through the m.sub.2 muscarinic acetylcholine receptor is coupled to pertussis toxin-sensitive G proteins and adenylyl cyclase activity. The gamma-aminobutyric type B receptor (GABA.sub.B) is similarly coupled to this signal transduction process, and both receptors are expressed in cerebellar granular neurons. Antisense oligonucleotides to the m.sub.2 receptor mRNA blocked completely the synthesis of this receptor within 3 days, and reduced the GABA.sub.B receptor by 40% within 6 days. It remains to be shown whether this latter effect was due to the conserved oligo sequence being present also in the yet uncloned GABA.sub.B receptor, or whether the delay effect was secondary to m.sub.2 inhibition Morrison R., ibid.!.
In view of the above-mentioned implication of cholinergic signals in the commitment and development of haematopoietic cells, it is an object of the present invention to provide for compounds and methods capable of diverting the process of cholinergic signalling, which may direct bone marrow stem cells into continued replication and re-orient their subsequent differentiation, both in culture and in vivo, into mononuclear cells of the hemopoietic and immune system.
Thus, the ChEs related antisense oligodeoxynucleotides of the present invention appear to be potent candidates for the modulation of bone marrow cells development described above. This adds to the effects of already characterized growth factors, such as the granulocyte colony stimulating factor (G-CSF), interleukin 3, 6 and 11, Lif (leukemia inducing factor) and the recently described stem cell factor, which interacts with the receptor produced from the C-kit protoncogene.